Many chemotherapy drugs target microtubules in order to inhibit mitosis and stop the proliferation of cancer cells. Some mitotic inhibitors alter microtubule assembly kinetics in order to stall or prevent microtubule growth, while others prevent the attachment of microtubules to the relevant mitotic machinery. However, the fundamental processes of microtubule growth and microtubule attachment to kinetochores remain poorly understood. The goal of this proposal is to elucidate how the molecular targets of mitotic inhibitors operate under normal conditions, and how anti-cancer therapeutics alter their mechanisms. The central approach utilized in this study is to reconstitute the mitotic machinery in vitro using purified recombinant proteins. This bottoms-up approach has the advantages of allowing for direct and complete control over experimental conditions, and of enabling single-molecule measurements to be made using powerful analytical techniques. In this proposal, a new microscopy technique called interferometric scattering microscopy (iSCAT) is refined and applied in order to answer mechanism questions centering around microtubules. iSCAT enables direct visualization of unlabeled microtubules at frame rates up to 50,000 frames per second, and can measure the position of proteins labeled with a 30-nm gold nanoparticle with 2-nm precision. This highly capable technique opens new doors for studying fast single-molecule kinetics. In the first aim of this proposal, a custom iSCAT microscope is constructed and used to discover how microtubule motors use ATP to produce force. In the second aim, iSCAT is used to track the fates of individual tubulin subunits within the microtubule lattice in order to quantitatively describe how microtubule dynamic instability is controlled in the absence and presence of anti-mitotic drugs. In the third aim, new advanced analytical techniques are used to study how microtubules attach to kinetochores during mitosis. Success in these aims will elucidate in quantitative detail how chemotherapy targets function, and will advance the treatment options for a broad array of cancers by guiding the development of new anti-mitotic drugs.